title: Identification, cloning, and characterization of two N-acetylgalactosamine-binding lectins from the albumen gland of Helix pomatia.
abstract: Helix pomatia agglutinin (HPA), the lectin from the albumen gland of the Roman snail, has been used in histochemical studies relating glycosylation changes to the metastatic potential of solid tumors. To facilitate the use of HPA in a clinical (diagnostic) setting, detailed analysis of the lectin, including cloning and recombinant production of HPA, is required. A combination of isoelectric focusing, amino acid sequence analysis, and cloning revealed two polypeptides in native HPA preparations (HPAI and HPAII), both consistent with GalNAc-binding lectins of the H-type family. Pairwise sequence alignment showed that HPAI and HPAII share 54% sequence identity whereas molecular modeling using SWISS-MODEL suggests they are likely to adopt similar tertiary structure. The inherent heterogeneity of native HPA highlighted the need for production of functional recombinant protein; this was addressed by preparing His-thioredoxin-tagged fusion products in Escherichia coli Rosetta-gami B (DE3) cells. The recombinant lectins agglutinated human blood group A erythrocytes whereas their oligosaccharide specificity, evaluated using glycan microarrays, showed that they predominantly bind glycans with terminal α-GalNAc residues. Surface plasmon resonance with immobilized GalNAc-BSA confirmed that recombinant HPAI and HPAII bind strongly with this ligand (K(d) = 0.60 nm and 2.00 nm, respectively) with a somewhat higher affinity to native HPA (K(d) = 7.67 nm). Recombinant HPAII also bound the breast cancer cells of breast cancer tissue specimens in a manner similar to native lectin. The recombinant HPA described here shows important potential for future studies of cancer cell glycosylation and as a reagent for cancer prognostication.
authors.affiliation: Department of Molecular and Applied Biosciences, School of Life Sciences, University of Westminster, 115 New Cavendish Street, London W1W 6UW, United Kingdom.
authors.forename: Anatoliy
authors.initials: A
authors.lastname: Markiv
authors.forename: Diluka
authors.initials: D
authors.lastname: Peiris
authors.forename: G Paul
authors.initials: GP
authors.lastname: Curley
authors.forename: Mark
authors.initials: M
authors.lastname: Odell
authors.forename: Miriam V
authors.initials: MV
authors.lastname: Dwek
mesh_terms: D000017:ABO Blood-Group System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000818:Animals; D003001:Cloning, Molecular; D005088:Exocrine Glands / Q000737:chemistry* / Q000378:metabolism; D006372:Helix, Snails / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D034781:Receptors, N-Acetylglucosamine / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D000017:ABO Blood-Group System; D034781:Receptors, N-Acetylglucosamine; D011994:Recombinant Proteins